A transcriptomic evaluation of the mechanism of programmed cell death of the replaceable bud in Chinese chestnut

Abstract Previous studies suggest that the senescence and death of the replaceable bud of the Chinese chestnut cultivar (cv.) “Tima Zhenzhu” involves programmed cell death (PCD). However, the molecular network regulating replaceable bud PCD is poorly characterized. Here, we performed transcriptomic profiling on the chestnut cv. “Tima Zhenzhu” replaceable bud before (S20), during (S25), and after (S30) PCD to unravel the molecular mechanism underlying the PCD process. A total of 5,779, 9,867, and 2,674 differentially expressed genes (DEGs) were discovered upon comparison of S20 vs S25, S20 vs S30, and S25 vs S30, respectively. Approximately 6,137 DEGs common to at least two comparisons were selected for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to interrogate the main corresponding biological functions and pathways. GO analysis showed that these common DEGs could be divided into three functional categories, including 15 cellular components, 14 molecular functions, and 19 biological processes. KEGG analysis found that “plant hormone signal transduction” included 93 DEGs. Overall, 441 DEGs were identified as related to the process of PCD. Most of these were found to be genes associated with ethylene signaling, as well as the initiation and execution of various PCD processes.


Introduction
Chinese chestnut (Castanea mollissima BL.) is an important nut-producing tree grown in temperate regions worldwide [1]. However, chestnut trees suffer reduced productivity over time due to the characteristics of branch growth. In chestnut trees, the apical bud of each existing fruiting branch will develop into a new fruiting branch in the following year, resulting in the majority of fruit development taking place along the peripheral crown. A notable exception, the spontaneous mutant chestnut cultivar (cv.) "Tima Zhenzhu," was first described in 1979. In "Tima Zhenzhu" trees, the apical bud (hereafter "replaceable bud") senesces and dies, resulting in a compact crown [2]. Research indicates that the death of the replaceable bud involves programmed cell death (PCD), as evidenced by the presence of certain markers of PCD, including chromatin condensation, nuclear degradation, DNA laddering, tonoplast invagination, vacuolar rupture, and autophagy, among others [1].
PCD, an orderly process of cellular suicide mediated by intracellular death programs [3], is crucial for normal tissue development and stress response [4]. In plants, developmental PCD regulated by internal factors occurs concomitantly during reproductive and vegetative development including cell death of the replaceable bud, root cap cells, nucellar tissue, and tapetum, as well as trichome formation, sex determination, endosperm, embryonic suspensor, xylogenesis, organ senescence, and aerenchyma formation [1,[5][6][7]. Across diverse PCD cases, a common regulatory network coordinated among PCD preparation, initiation, effectors, and degradation has been documented in the regulatory mechanism of plant PCD [7]. Cellular preparation for PCD is coordinated primarily by transcriptional regulation of hormone signaling, most commonly of the ethylene signaling pathway [8][9][10][11]. Transcription factors (TFs) link phytohormone signaling to PCD regulation [12]. Downstream of hormone signaling, PCD can be triggered in specific cell types by diverse cellular events such as changes in intracellular Ca 2+ concentration, buildup of reactive nitrogen species and reactive oxygen species (ROS), activation of protein kinases, acidification of the cytoplasm, and modification of the cytoskeleton [13]. In addition, a plethora of hydrolytic enzymes, including nucleases and proteases, have been suggested as putative PCD executors [5,6,14].
In the present study, we performed transcriptomic profiling of the replaceable bud of Chinese chestnut cv. "Tima Zhenzhu" before (S20), during (S25), and after (S30) PCD. This study aimed to (i) investigate the expression patterns of the differentially expressed genes (DEGs) as well as their functions and metabolism pathways, and (ii) elucidate the regulatory and signaling pathways related to replaceable bud PCD in chestnut cv. "Tima Zhenzhu." The results of this work will not only bolster our understanding of the molecular mechanism of replaceable bud PCD but may also provide a relevant resource for the future genetic improvement of chestnuts.

Plant materials
Chestnut cv. "Tima Zhenzhu" (C. mollissima BL.) trees were kept at the Changli Institute of Pomology, Academy of Agricultural and Forestry Sciences, Hebei Province, China (118°51′ E, 39°53′ N). Tree age 12 years, row spacing 4 m × 4 m. A total of 15 trees with normal growth and no pests and diseases were selected. Based on previous research results, replaceable buds of the first three nodes of the top of the bearing branches with the same thickness and length were collected at 20 (S20), 25 (S25), and 30 (S30) days after flowering, corresponding to the developmental stage before, during, and after PCD . At S20, the buds are green and display a normal phenotype; at S25, the buds are yellow-green but no abscission layer formed; at S30, the buds are brown-yellow and display a partial abscission layer ( Figure 1). Three biological replicates were collected at each phase, and each biological replicate contains 60 buds from 5 trees (a total of 15 trees).

Identification and functional annotation of DEGs
Raw sequence data were filtered by removing adapter sequences and low-quality reads. HISAT2 software [15] was utilized to map clean reads to the C. mollissima reference genome [16]. StringTie software [17] was utilized to quantify gene expression as fragments per kilobase of exon per million mapped reads (FPKM) values. Genes were considered as "expressed" if their FPKM was >0. DESeq2 software [18] was utilized to discover DEGs between the S20, S25, and S30 time points, with a threshold false discovery rate (FDR) set at <0.01 and |log2FC| > 1. Finally, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) was utilized for both analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and annotation of gene ontology (GO), as implemented in BMK Cloud provided by Biomarker Technologies Company (Beijing, China).

Protein-protein interaction network construction
Protein-protein interactions were queried using the STRING database (https://string-db.org/cgi). The proteins encoded by DEGs putatively involved in replaceable bud PCD were networked using their respective tobacco homologs. The Cytoscape software [19] was utilized to visualize the resulting protein-protein interaction network.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation
An RNAqueous Total RNA Isolation Kit (Solebo Technology, China) was utilized to extract total RNA from the replaceable bud before (S20), during (S25), and after (S30) PCD. The HiScript II Q RT SuperMix for qPCR (Yisheng Biotechnology, China) was utilized to synthesize cDNA from the extracted RNA, with Actin serving as the internal reference gene. All qRT-PCR primers can be found in Table S7. A LightCycler 480II Real-Time PCR Detection System (Roche, Switzerland) was utilized to perform qRT-PCR. The reaction mixture contained 2 µL of the cDNA template, 10 μL of the ChamQ SYBR qPCR Master Mix, 6.8 μL of deionized water, 0.4 μL of ROX Reference Dye2, 0.4 µL of the forward primers, and 0.4 µL of the reverse primers. The amplification program was conducted as follows: 95°C for 5 min; 40 cycles of 95°C for 10 s, and 60°C for 30 s. To ensure data reproducibility, three independent experiments were conducted. The 2 −△△Ct method [13] was utilized to quantify the levels of relative gene expression.
3.2 Global transcriptional changes before, during, and after PCD in replaceable buds of chestnut cv. "Tima Zhenzhu" In order to identify DEGs, a stringent threshold of FDR < 0.01 and |log 2 FC| > 1 was used. In total, 5,779 DEGs were identified in S20 vs S25, 9,867 in S20 vs S30, and 2,674 in S25 vs S30 ( Figure 3). The large number of DEGs (10,870 in total; Table S1) suggested that replaceable bud PCD may be a complex process regulated by an extensive population of genes. Moreover, we found a significantly greater number of DEGs in S20 vs S30, compared to the other two timepoint comparisons, indicating that gene expression in replaceable bud PCD is highly variable across time. Clear differences were found in global gene expression patterns between the three time points (Figure 3). In general, the amount of both down-and upregulated genes was large in all comparisons, although the amount of down-regulated genes tended to be slightly higher ( Figure 3).

qRT-PCR validation of the transcriptomic data
Nine PCD-related DEGs were chosen for further qRT-PCR analysis to confirm their expression levels during replaceable bud PCD of chestnut cv. "Tima Zhenzhu." Overall, the RNA-seq results were largely in agreement with the qRT-PCR results (Figure 8), suggesting that our transcriptomic analysis was reasonable and accurate.

Role of the ethylene signaling in replaceable bud PCD of chestnut cv. "Tima Zhenzhu"
Phytohormone signal transduction is likely the prevailing mechanism responsible for the upstream regulation of PCD processes [7]. For example, ethylene is responsible for leaf senescence and root deterioration in maize [10,11]; auxin is responsible for developmentally regulated PCD in lace plants [20]; GA is related to aleuronal PCD [21]; and the combination of auxin and BR signal transduction may indirectly mediate PCD to regulate pericarp thickness in the sweet corn [22]. Here, KEGG analysis also indicated that the "plant hormone signal transduction" (ko04075) pathway was particularly enriched. Several key components of the ethylene signaling pathway were found to be upregulated at S25, including ERS1 (EVM0016645) and EIN3 (EVM0013705, EVM0023854, and EVM0025637) ( Figure 6). In particular, EIN3 exerts control over cellular senescence and death through its involvement in a trifurcate feed-forward pathway [23]. Within the EIN2-EIN3-NAC regulatory cascade controlling leaf senescence-associated PCD, EIN3 acts to transcriptionally activate NAC TFs (ORE1 and AtNAP), which themselves act to positively regulate leaf senescence [24]. Additionally, EIN3 functions as a direct repressor of miR164 [23], which acts to post-transcriptionally negatively regulate ORE1. In order to efficiently regulate leaf senescence, EIN3 appears to simultaneously regulate the expression of both ORE1 and its particular negative regulator (miR164). By analogy, the upregulation of EIN3 at S25 likely promotes replaceable bud PCD. Taken together, these findings suggest that ethylene signaling is activated during preparation for PCD in replaceable buds of chestnut cv. "Tima Zhenzhu." In our previous study, we reported that the ethylene content was higher in replaceable buds at S25 than at S20 [25]. Our present study bolsters this observation, as we found that genes (encoding ACO and ACS) related to ethylene biosynthesis were consistently upregulated at S25 (Table S6). We identified additional DEGs involved in other hormone signaling pathways, suggesting that several phytohormones may interact during replaceable bud PCD. For example, both GA and ABA antagonistically regulate aleuronal PCD [5]. Additionally, leaf senescence is promoted by a combination of ethylene, SA, and ABA, while this process is delayed by a combination of auxin, cytokinin, and GA [26,27]. As shown in Table S5 and Figure 6, DEGs involved in ethylene signaling, such as MAPK (EVM0010215), ERS1 (EVM0016645), EIN3 (EVM0013705 and EVM0025637), and ERF1B (EVM0030131 and EVM0003409), were upregulated at S25 vs S20. Contrarily, DEGs involved in the auxin pathway, such as AUX28 (EVM0014325), SAUR-like auxin-responsive proteins (EVM0004337, EVM0007905, EVM0019733, and EVM0021087), and auxin-responsive proteins (e.g., EVM0003597, EVM0004089, EVM0005109, EVM0007851, EVM0017536, and EVM0017744), or the cytokinin pathway, such as AHP5 (EVM0015700) and two-component response regulator ARR family members (EVM0008810, EVM0017362, EVM0020552, and EVM0022760), were downregulated at this same time point. In our previous study, we reported that auxin and cytokinin content were significantly lower in replaceable buds at S25 than at S20 [25]. Taken together, it appears that replaceable bud PCD of chestnut cv. "Tima Zhenzhu" is primarily controlled by high ethylene and low auxin and cytokinin signaling, suggesting that ethylene may antagonistically interact with auxin and cytokinin in this process.

PCD initiation in replaceable buds of chestnut cv. "Tima Zhenzhu"
Several TFs may act as bridges linking phytohormone signaling with PCD regulation [33]. As an example, ORESARA1 (ANAC092, a NAC TF) regulates leaf senescence both downstream of ethylene signaling and upstream of senescenceinducing genes, including NAC TFs such as BIFUNCTIONAL NUCLEASE 1 (BFN1) [34]. In this study, we discovered a large number of DEGs encoding TF homologs. These results suggest that several TFs may act to ensure the initiation of bud PCD of chestnut cv. "Tima Zhenzhu." Cytochrome c plays a part in the PCD signaling network, with the upregulation of both cytochrome c oxidase and cytochrome c being an early event during the process of PCD [35]. The process of PCD in several plants is accompanied by the dispensation of mitochondrial cytochrome c, including tapetal PCD in sunflowers and terminally differentiated suspensor PCD in runner bean cotyledons [36,37]. Here, we found that the expression levels of genes encoding cytochrome c (EVM0024076) and cytochrome c oxidases (EVM0029309, EVM0032493, EVM0012209, EVM0004365, EVM0022679, and EVM0012699) were notably upregulated at S25 compared to S20 (Table S6). Therefore, we suggest that cytochrome c and cytochrome c oxidases may modulate replaceable bud PCD initiation in chestnut cv. "Tima Zhenzhu." Substantial evidence indicates that Ca 2+ , a universal second messenger, is crucial for PCD regulation in plants. Cytoplasmic Ca 2+ -influx from vacuoles and endoplasmic reticulum is an early event during PCD [38]. Specifically, Ca 2+ -permeable channels, Ca 2+ sensors including calcineurin B-like proteins (CBLs), calmodulins (CAMs), and CDPKs are necessary for Ca 2+ signal transduction and PCD [39]. The activities of many enzymes and other proteins associated with various PCDs are under the control of Ca 2+ /CAM or Ca 2+ /CBL complex such as the Ca 2+ -dependent endonucleases and hydrolytic enzymes [40]. CDPK3 is a positive regulator of LCB (sphingoid long-chain bases)-mediated PCD in Arabidopsis [41]. In the present research, a total of 36 DEGs were found to be associated with Ca 2+ signaling, consisting of 4 Ca 2+ channels, 9 CDPKs, 9 calcineurin B-like proteins, and 14 calmodulins. Most of these DEGs exhibited similar expression patterns with significantly higher levels at S25 compared to S20 (Table S6), suggesting that cascade events are likely to contribute to the calcium signaling. Moreover, in our previous study, increased calcium levels were observed in the replaceable bud cells undergoing PCD in chestnut cv. "Tima Zhenzhu" [42]. Therefore, it is reasonable to infer that the calcium-dependent signaling cascade should be related to modulating the replaceable bud PCD initiation in chestnut cv. "Tima Zhenzhu." Furthermore, in tomatoes, Ca 2+ -CBL10/ CIPK6 complex promotes the accumulation of ROS by activating the respiratory burst homolog RBOH and hence regulates the process of PCD [43]. Similar results were found in the present research: in the constructed protein-protein interaction network (Figure 8), A0A1S3Y4S2, an RBOH, showed strong interactions with five CDPKs, and both RBOH and CDPKs exhibited upregulated expression with significantly higher levels at S25 and S30 compared to S20, these results suggest that the CDPKs may promote the accumulation of ROS by activating RBOH, which may also be involved in the downstream cascade of Ca 2+ regulatory pathway in replaceable bud PCD of chestnut cv. "Tima Zhenzhu." 4.3 PCD execution in replaceable buds of chestnut cv. "Tima Zhenzhu" The reception of specific signal triggers initiates PCD execution and cellular corpse clearance, activating and releasing myriad hydrolytic enzymes, including various nucleases and proteases [33]. Our previous study found no evidence of DNA degradation at S20, with some DNA degradation at S25 and heavy DNA degradation at S30 [1]. Here, we found two DEG encoding nucleases, including endonuclease V isoform X1 (EVM0021509) and endonuclease 1-like isoform X2 (EVM0022275), which were significantly upregulated at S25 and S30 compared to S20 (Figure 8; Table S6), may promote the DNA degradation of bud PCD in chestnut cv. "Tima Zhenzhu." We also found a variety of DEGs (upregulated at S25 and S30 compared to S20) encoding proteases, including cysteine proteinases RD21A-like, metacaspase-9-like, aspartic proteinases, vacuolar-processing enzyme-like, senescence-associated proteins, endoglucanases, pectinesterase, xyloglucan endotransglucosylase/hydrolases, xyloglucan galactosyltransferase, and exosome complex component RRP45A-like. Previous studies on homologs of our identified cysteine proteinases RD21A-like, metacaspase-9-like, aspartic proteinases, vacuolar-processing enzyme-like, and senescence-associated proteins suggest that these enzymes are crucial for effective PCD by degrading many essential cellular targets [14,19,33,44]. Endoglucanases, pectinesterases, xyloglucan endotransglucosylase/hydrolases, and xyloglucan galactosyltransferases may act to recycle carbohydrates as cells undergo PCD. Finally, homologs of complex exosome component RRP45A-like likely participate in DNA degradation [13]. Autophagy is a metabolic mechanism whereby cytoplasmic substances and organelles are degraded by lysosomes or vacuoles and is a requirement for efficient PCD in many plant systems [45]. Autophagic cell death is indicated by several distinct morphological features in plants, such as an increase in vacuolar and cellular size, movement of organelles into the vacuole for destruction, and subsequent cell death resulting from vacuolar lysis [46,47]. Autophagy plays both pro-survival and pro-death roles in plant PCD [47]. The core autophagy mechanism is organized by an evolutionarily conserved ATG (AuTophaGy-related) gene population [46]. In our previous study, we found that during replaceable bud PCD of chestnut cv. "Tima Zhenzhu," small vacuoles in the cytoplasm fused to form a large vacuole, resulting in the eventual degradation of other CCs [1]. Here, in accordance with this observation, we found that "regulation of autophagy" pathway genes were upregulated at S25 and S30 compared to S20, including autophagy-related proteins 8f, 16, 8i-like, 8C-like, 3 and 13a (EVM0027149, EVM0009961, EVM0003991, EVM0004768, EVM0015502, EVM0002553, EVM0027459, and EVM0017527), cysteine protease ATG4-like (EVM0001368), ubiquitin-like modifieractivating enzyme ATG7 (EVM0017393), ubiquitin-like protein ATG12 (EVM0018088), and CBL-interacting serine/threonineprotein kinase 1-like and 6-like (EVM0001436 and EVM0010887) (Table S9). Additionally, the highly selective autophagy of soluble proteins is mediated by the ESCRT (endosomal sorting complex required for transport) complex, which requires vacuolar protein sorting (VPS) [48]. We found six DEGs (five up-and one downregulated at S25 and S30 compared to S20) encoding VPS-associated proteins (A0A1S4DPC1, A0A1S4DKE1, A0A1S4CYV6, A0A1S4BA99, A0A1S3WXN7, and A0A1S4DLQ1) which interacted with each other and with the network of autophagy-related proteins (A0A1S3Y2M7, ATG8e, A0A1S4A7C8, ATG4, A0A1S4D5L9, ATG5, A0A1S4C6J8, ATG9, ATG2, A0A1S4DLP2, ATG13a, and A0A1S4BPT6) ( Figure 7; Table S8). Autophagic processes are necessary for the timely progression of replaceable bud PCD of chestnut cv. 'Tima Zhenzhu,' and that replaceable bud PCD may depend on VPS-associated proteins.

Conclusions
We identified the DEGs and signaling pathways responsible for regulating replaceable bud PCD in chestnut cv. "Tima Zhenzhu" through transcriptomic profiling. Based on our cumulative results, we offer a hypothetical model of replaceable bud PCD consisting of three overlapping processes ( Figure 9). First, ethylene signaling is activated during preparation for PCD in order to regulate the activity of downstream targets. Next, during PCD initiation, the upregulation of several TFs (including MYB, MADS-box, bHLH, and NAC TFs) induces an increase in cytochrome c expression and the cytosolic Ca 2+ content, activating the Ca 2+dependent signaling cascade. Finally, during PCD execution, the process of autophagy and several proteases (i.e., cysteine proteinases RD21A-like, metacaspase-9-like, vacuolar-processing enzyme-like, and senescence-associated proteins) work synergistically to clear the cell of CCs. When this process is complete, the replaceable bud senesces and dies. This hypothetical model will bolster our understanding of the molecular mechanism of bud PCD. In the later stage, we need to do further research on gene function and gene interaction through transgenic systems, yeast two-hybrid systems, and other technical methods.
Acknowledgements: The authors would like to thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript. Author contributions: YG, SHZ, YL, XFZ, GPW, HL, SYL, and JL contributed to the study conception and design. Material preparation, data collection and analysis were performed by YG, and XFZ. The first draft of the manuscript was written by YG, and GPW commented on previous versions of the manuscript. All authors read and approved the final manuscript.